DESCRIPTION (adapted from the applicant's description): Plants can detect specific pathogens and activate defense mechanisms. This proposal is aimed at understanding how pathogen detection is accomplished, defining the specificity determinants, and identifying components involved in translating the detection into a disease resistance response. The research addresses these three major questions using a combination of genetics, molecular biology and protein biochemistry. The investigator initially identified and isolated an Arabidopsis disease resistance "R" gene (RPS5) that mediates specific recognition of the avirulence protein (avrphB) secreted by the bacterial pathogen Pseudomonas syringae. RPS5 belongs to the largest class of plant resistance (R) genes encoding cytosolic proteins defined by the presence of a putative nucleotide binding site (NBS) and leucine rich repeats (LRRs). NBS-LRR R proteins are believed to function as cytoplasmic receptors that directly bind pathogen-derived ligands via the LRR (protein interaction) domain. However, direct proof of this receptor-ligand model is still lacking. It is equally plausible that NBS-LRR R proteins specifically interact with other plant proteins, which are themselves the targets of pathogen virulence factors; pathogens would thus detect a change in conformation of these target proteins. A specific goal of the proposed research is to identify the mechanism by which RPS5 detects the bacterial avrPphB protein. Extensive mutant screens have uncovered three plant genetic loci required for RPS5-mediated disease resistance: PBS1, PBS2 and PBS3. The PBS1 protein is specifically required for the RPS5 pathway, and thus could potentially function as the target of avrPphB and the ligand for RPS5. Because recognition of avrPphB requires both PBS1 and RPS5, it is proposed to test whether the PBS1 protein interacts with RPS5 and/or avrPphB. The gene encoding the PBS1 protein has been cloned and exhibits significant homology to a large family of plant-specific protein kinases (Pto and Pti kinases) that participate in recognition of the avrPto protein of P. syringae. With the recent identification of PBS1 as a putative kinase, the investigator will test whether RPS5 is a substrate for phosphorylation by PBS1. In addition, with all three cloned genes in hand, specific interactions between PBS1, avrPphB and RPS5 will be evaluated using various assays. The mechanisms by which RPS5 itself induces defense responses will also be investigated. An agrobacterium-based transient expression assay will be used to identify dominant negative and constitutively activated alleles of RPS5. Such alleles will provide insight into how RPS5 signaling is regulated. Wild-type and mutant RPS5 proteins will be used in protein-protein interaction assays with candidate downstream effectors. Examples of the latter include NDR1, PBS2 and PBS3. The NDR1 gene has already been cloned, and cloning of PBS2 and PBS3 will be pursued. If stable complexes are observed, complexes will be purified and protein components will be identified using mass spectrometry. These experiments will advance understanding of how interactions of plant R genes and pathogen avr genes lead to disease resistance.